The Role of Dll1 in Specifying the Molecular and Functional Status of Horizontal Basal Cells

项目摘要 近四分之三的美国人在80岁之前会经历嗅觉的功能性衰退，这可能会 危及营养状况、身体安全，并对生活质量产生负面影响。不幸的是，有一些 目前还没有有效的治疗方法来逆转老年性营养不良。这种感觉缺陷可以归因于 老年人嗅觉上皮(OE)内无嗅觉的病理恶化区域 感觉神经元。神经元区域持续存在是因为水平基底细胞(HbCs)，即OE的储备居民 能够产生所有OE细胞类型的干细胞群体仍然处于令人沮丧的休眠状态。正在激活 因此，在老年人类OE中持续存在的多潜能HBCs代表了一种潜在的强大方法 扭转气味损失。我们的实验室以前已经证明了切迹的条件敲除(CKO) HbC中受体Notch1导致HbC转录因子(Tf)和MASTER表达降低 调节HBC状态的P63，并沉淀自发的HBC激活。然而，要巧妙地操纵 目前已知的唯一调节HbCs分子和功能状态的信号通路，我们需要一个 对Notch信号及其在指定HBC状态中的作用有更深入的了解。这种需要是由于固有的 Notch信号通路的复杂性。这一复杂性的一个方面包括能够使用单个凹槽 受体类型与多个配体相互作用，每个配体都可以不同地影响细胞分化。在……里面 HbCs表达的OE，Notch1可能与几种Notch配体相互作用，包括Dll1，即 HBCs和Jagged1也表达，定位于神经胶质样支持细胞。正如我的数据显示的那样 HBc表达的DLL1和Notch1确实存在相互作用，目的1将建立DLL1对功能和功能的S效应 通过(I)定量Hes1，一种Notch信号转导途径，研究未损伤OE中休眠的HbCs(DHbCs)的分子同一性。 原代小鼠HBC培养中DLL1 CKO后Tf和p63的依赖表达。我还将(Ii)利用 荧光激活细胞分选技术定量检测HbCKO后Hes1和p63的体内表达 Dll1.为了探索DLL1在HBC中的长期功能作用，我将(III)量化DLL1的数量和身份 D111的HBC CKO后的HBC衍生后代。因为Notch配体-受体的功能输出 相互作用可以在组织形态发生过程中动态调节，Aim 2将阐明 Dll1在直接OE诱导的HBC介导的OE再生模型中对活化HbCs(AHbCs)的作用 受伤。为了做到这一点，我将(I)进行流式细胞术来关联，与细胞分辨率之间的关系 HBC活性状态和DLL1表达。我还将(Ii)询问Dll1对功能和分子的影响 在OE后立即通过病毒转导在HBCs中过表达Dll1对再生有影响 受伤。根据NIDCD的使命，该项目及其多学科方法将有助于设计医疗 治疗性地激活HbCs并可控制地再生OE以逆转年龄相关的治疗 性欲减退。因此，我将通过这一奖学金为开始内科科学家的职业生涯做好准备。

Project Summary A functional decline in smell, which is experienced by nearly three-quarters of Americans by age 80, can endanger nutritional status, physical safety, and negatively impact quality of life. Unfortunately, there are currently no effective treatments to reverse age-related hyposmia. This sensory deficit can be attributed to pathologically deteriorated regions within aged human olfactory epithelium (OE) that are void of olfactory sensory neurons. Aneuronal regions persist because horizontal basal cells (HBCs), the OE's reserve resident stem cell population that is capable of giving rise to all OE cell types, remain frustratingly dormant. Activating multipotent HBCs that endure within aged human OE, therefore, represents a potentially powerful approach to reverse smell loss. Our laboratory has previously demonstrated that conditional knock out (cKO) of the Notch receptor, Notch1, in HBCs leads to decreased HBC expression of the transcription factor (TF) and master regulator of HBC status, p63, and precipitates spontaneous HBC activation. However, to deftly manipulate the only signaling pathway currently known to regulate the molecular and functional status of HBCs, we require a deeper understanding of Notch signaling and its role in specifying HBC status. This need is due to the inherent complexities of the Notch signaling pathway. One aspect of this intricacy includes the ability for a single Notch receptor type to interact with multiple ligands, each of which can differentially impact cellular differentiation. In the OE, Notch1 expressed by HBCs can potentially interact with several Notch ligands, including Dll1 that is also expressed by HBCs and Jagged1 localized to glial-like Sustentacular cells. As my data demonstrates that HBC-expressed Dll1 and Notch1 do indeed interact, Aim 1 will establish Dll1's effect on the functional and molecular identity of dormant HBCs (dHBCs) within uninjured OE by (i) quantifying Hes1, a Notch signaling- dependent TF, and p63 expression following Dll1 cKO in primary mouse HBC cultures. I will also (ii) utilize fluorescence activated cell sorting to quantify in vivo HBC expression of Hes1 and p63 following HBC cKO of Dll1. To probe the longer-term functional role of Dll1 in HBCs, I will (iii) quantify the number and identity of HBC-derived progeny following HBC cKO of Dll1. Because the functional output of Notch ligand-receptor interactions can be dynamically regulated during tissue morphogenesis, Aim 2 will elucidate the influence that Dll1 exerts on activated HBCs (aHBCs) in a model of HBC-mediated OE regeneration elicited by direct OE injury. To do so, I will (i) perform flow cytometry to correlate, with cellular resolution, the relationship between HBC activity status and Dll1 expression. I will also (ii) interrogate the functional and molecular impact that Dll1 has on regeneration by overexpressing Dll1 in HBCs via viral transduction in the immediate aftermath of OE injury. In line with NIDCD's mission, this project and its multidisciplinary approach will aid the design of medical treatments that therapeutically activate HBCs and controllably regenerate the OE to reverse age-related hyposmia. As a result, I will be well prepared by this fellowship to embark on a career as a physician-scientist.